Bio-Rad ProteinChip Antibody Capture Kit User Manual
ProteinChip®Antibody Capture Kit
Instruction Manual
Catalog #K100-0005
For technical support,
call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD
(1-800-424-6723).
Introduction
Traditional immunoassay-based protein detection methods are often difficult to
develop and are complicated by antibodies cross-reacting and binding
nonspecifically to a variety of proteins and peptides. The ProteinChip antibody (Ab)
capture kit enables unambiguous species-specific protein identification and epitope
discovery experiments on one platform, and eliminates the need to develop a
sandwich assay. Using the components provided in the kit, antibodies are bound to
a ProteinChip array to detect antigen(s) of interest from crude samples derived from
in vivo or in vitro experimental models. Once an antibody-antigen complex has been
formed on the ProteinChip array, researchers can use the ProteinChip array for two
unique purposes: 1) to identify proteins bound to the antibody on the basis of
molecular weight using the ProteinChip SELDI reader, or 2) to conduct epitope
discovery experiments by proteolytically cleaving proteins captured on ProteinChip
arrays (on-chip proteolysis) and then using SELDI to highlight the peptide(s)
associated with the epitope, streamlining the process of sequencing the protein
binding site via tandem mass spectrometry (MS/MS).
Fig. 1. Principles of the ProteinChip antibody capture kit. Protein G is covalently bound to the ProteinChip
PG20 array surface. An optional crosslinking step can be performed to crosslink IgG antibodies to protein G.
© 2007 Bio-Rad Laboratories, Inc.
The key component of this kit is the ProteinChip PG20 array (ProteinChip PS20 array
precoupled with recombinant protein G (Pierce). Any antibody capable of binding
protein G can be used with the kit (see appendix for details). The kit also contains
positive (TNF-α antigen-antibody pair) and negative (bovine IgG) controls and a
crosslinking reagent to bind antibodies covalently to ProteinChip PG20 arrays.
Materials
Materials Included
n
TNF-α antibody (0.2 mg/ml anti-human TNF-α goat polyclonal antibody in
phosphate buffered saline (PBS), 20 µl
n
TNF-α antigen* (1.0 µM recombinant human TNF-α antigen in 0.1% bovine
serum albumin (BSA)/PBS), 10 µl
n
Bovine IgG (lyophilized), 750 µg
n
Wash buffer (0.5% Triton X-100 in PBS); 2 bottles, 120 ml each
n
PBS; 2 bottles, 120 ml each
n
1x PBS; 1 bottle, 4 ml**
n
ProteinChip PG20 arrays, 6
n
Bis(sulfosuccinimidyl) suberate (BS3) crosslinking reagent (0.5 mg/vial), 2
n
Deactivation buffer (0.5 M ethanolamine in PBS, pH 8.0), 4 ml
n
ProteinChip sinapinic acid (SPA) energy absorbing molecules (EAMs), 5 mg
n
Instruction manual
* Contains equimolar amounts of TNF with and without N-terminal methionine, yielding a double peak.
** The 4 ml PBS should be used to dilute the bovine IgG, antibody, and antigen, reconstitute the
crosslinking reagent, and deposit to the spots of the ProteinChip array when needed.
© 2007 Bio-Rad Laboratories, Inc.
M
aterials Needed but Not Included
n
15 ml conical tubes
n
Rocking platform
n
Humidity chamber*
n
Lab timer
n
Ice bucket and ice
n
Microcentrifuge tubes
n
Calibrated pipets**
n
99+% trifluoroacetic acid (TFA)
Optional Materials
n
ProteinChip cassette-compatible bioprocessor (catalog #C50-30011)
n
MicroMix 5 plate and tube shaker*** (Diagnostic Products Corporation)
n
Sonicating water bath
Shipping and Storage
Table 1. Shipping and storage conditions for ProteinChip antibody capture kit components.
Item Shipping Storage
Anti-human TNF-α Dry ice –20°C (stable 6 months)
antibody 4°C (stable 2 weeks)
Recombinant human Dry ice –20°C (stable 6 months)
α antigen 4°C (stable 2 weeks)
TNF-
Bovine IgG Dry ice –20°C
Crosslinking reagent Dry ice –20°C
ProteinChip PG20 arrays Ambient with desiccant 4°C with desiccant
Wash buffer Ambient 4°C
PBS Ambient 4°C
Deactivation buffer Ambient 4°C
HEPES buffer Ambient 4°C
ProteinChip SPA EAMs Ambient Ambient or 4°C
* See the ProteinChip SELDI
humidity chamber if one is not r
System Applications Guide for detailed instructions on how to create a
eadily available.
** Calibrated pipets will yield more reproducible results.
*** When using a ProteinChip bioprocessor, a MicroMix 5 is recommended.
© 2007 Bio-Rad Laboratories, Inc.
Loading...